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T-type Ca 2 + channels (TTCCs) are expressed in the fetal heart and then disappear from ventricular myocytes after birth. The hypothesis examined in this study was the α1G TTCCs' influence in myocyte maturation and their rapid withdrawal from the cell cycle after birth. Methods Cardiac myocytes were isolated from neonatal and adult wild type (WT), α1G −/− and α1G over expressing (α1GDT) mice. Bromodeoxyuridine (BrdU) uptake, myocyte nucleation, cell cycle analysis, and T-type Ca 2 + currents were measured. Results All myocytes were mono-nucleated at birth and 35% of WT myocytes expressed functional TTCCs. Very few neonatal myocytes had functional TTCCs in α1G −/− hearts. By the end of the first week after birth no WT or α1G −/− had functional TTCCs. During the first week after birth about 25% of WT myocytes were BrdU + and became bi-nucleated. Significantly fewer α1G −/− myocytes became bi-nucleated and fewer of these myocytes were BrdU +. Neonatal α1G −/− myocytes were also smaller than WT. Adult WT and α1G −/− hearts were similar in size, but α1G −/− myocytes were smaller and a greater % were mono-nucleated. α1G over expressing hearts were smaller than WT but their myocytes were larger. Conclusions The studies performed show that loss of functional TTCCs is associated with bi-nucleation and myocyte withdrawal from the cell cycle. Loss of α1G TTCCs slowed the transition from mono- to bi-nucleation and resulted in an adult heart with a greater number of small cardiac myocytes. These results suggest that TTCCs are involved in the regulation of myocyte size and the exit of myocytes from the cell cycle during the first week after birth.

Rationale: Catecholamines increase cardiac contractility, but exposure to high concentrations or prolonged exposures can cause cardiac injury. A recent study demonstrated that a single subcutaneous injection of isoproterenol (ISO; 200 mg/kg) in mice causes acute myocyte death (8%–10%) with complete cardiac repair within a month. Cardiac regeneration was via endogenous cKit + cardiac stem cell–mediated new myocyte formation. Objective: Our goal was to validate this simple injury/regeneration system and use it to study the biology of newly forming adult cardiac myocytes. Methods and Results: C57BL/6 mice (n=173) were treated with single injections of vehicle, 200 or 300 mg/kg ISO, or 2 daily doses of 200 mg/kg ISO for 6 days. Echocardiography revealed transiently increased systolic function and unaltered diastolic function 1 day after single ISO injection. Single ISO injections also caused membrane injury in ≈10% of myocytes, but few of these myocytes appeared to be necrotic. Circulating troponin I levels after ISO were elevated, further documenting myocyte damage. However, myocyte apoptosis was not increased after ISO injury. Heart weight to body weight ratio and fibrosis were also not altered 28 days after ISO injection. Single- or multiple-dose ISO injury was not associated with an increase in the percentage of 5-ethynyl-2′-deoxyuridine–labeled myocytes. Furthermore, ISO injections did not increase new myocytes in cKit +/Cre ×R-GFP transgenic mice. Conclusions: A single dose of ISO causes injury in ≈10% of the cardiomyocytes. However, most of these myocytes seem to recover and do not elicit cKit + cardiac stem cell–derived myocyte regeneration.

Cardiac hypertrophy occurs as an adaptive response to increased workload to maintain cardiac function. However, prolonged cardiac hypertrophy causes heart failure, and its mechanisms are largely unknown. Here we show that cardiac angiogenesis is crucially involved in the adaptive mechanism of cardiac hypertrophy and that p53 accumulation is essential for the transition from cardiac hypertrophy to heart failure. Pressure overload initially promoted vascular growth in the heart by hypoxia-inducible factor-1 (Hif-1)-dependent induction of angiogenic factors, and inhibition of angiogenesis prevented the development of cardiac hypertrophy and induced systolic dysfunction. Sustained pressure overload induced an accumulation of p53 that inhibited Hif-1 activity and thereby impaired cardiac angiogenesis and systolic function. Conversely, promoting cardiac angiogenesis by introducing angiogenic factors or by inhibiting p53 accumulation developed hypertrophy further and restored cardiac dysfunction under chronic pressure overload. These results indicate that the anti-angiogenic property of p53 may have a crucial function in the transition from cardiac hypertrophy to heart failure.

Cardiac myosin binding protein C (cMyBP-C) has three phosphorylatable serines at its N terminus (Ser-273, Ser-282, and Ser-302), and the residues' phosphorylation states may alter thick filament structure and function. To examine the effects of cMyBP-C phosphorylation, we generated transgenic mice with cardiac-specific expression of a cMyBP-C in which the three phosphorylation sites were mutated to aspartic acid, mimicking constitutive phosphorylation (cMyBP-C AllP+ ). The allele was bred into a cMyBP-C null background (cMyBP-C (t/t) ) to ensure the absence of endogenous dephosphorylated cMyBP-C. cMyBP-C AllP+ was incorporated normally into the cardiac sarcomere and restored normal cardiac function in the null background. However, subtle changes in sarcomere ultrastructure, characterized by increased distances between the thick filaments, indicated that phosphomimetic cMyBP-C affects thick–thin filament relationships, and yeast two-hybrid data and pull-down studies both showed that charged residues in these positions effectively prevented interaction with the myosin heavy chain. Confirming the physiological relevance of these data, the cMyBP-C AllP+:(t/t) hearts were resistant to ischemia–reperfusion injury. These data demonstrate that cMyBP-C phosphorylation functions in maintaining thick filament spacing and structure and can help protect the myocardium from ischemic injury.

New neurons generated in the adult dentate gyrus are constantly integrated into the hippocampal circuitry and activated during encoding and recall of new memories. Despite identification of extracellular signals that regulate survival and integration of adult-born neurons such as neurotrophins and neurotransmitters, the nature of the intracellular modulators required to transduce those signals remains elusive. Here, we provide evidence of the expression and transcriptional activity of nuclear factor of activated T cell c4 (NFATc4) in hippocampal progenitor cells. We show that NFATc4 calcineurin-dependent activity is required selectively for survival of adult-born neurons in response to BDNF signaling. Indeed, cyclosporin A injection and stereotaxic delivery of the BDNF scavenger TrkB-Fc in the mouse dentate gyrus reduce the survival of hippocampal adult-born neurons in wild-type but not in NFATc4 −/− mice and do not affect the net rate of neural precursor proliferation and their fate commitment. Furthermore, associated with the reduced survival of adult-born neurons, the absence of NFATc4 leads to selective defects in LTP and in the encoding of hippocampal-dependent spatial memories. Thus, our data demonstrate that NFATc4 is essential in the regulation of adult hippocampal neurogenesis and identify NFATc4 as a central player of BDNF–driven prosurvival signaling in hippocampal adult-born neurons.

Forkhead Box M1 (Foxm1) is a transcription factor essential for organ morphogenesis and development of various cancers. Although complete deletion of Foxm1 in Foxm1(-/-) mice caused embryonic lethality due to severe abnormalities in multiple organ systems, requirements for Foxm1 in cardiomyocytes remain to be determined. This study was designed to elucidate the cardiomyocyte-autonomous role of Foxm1 signaling in heart development. We generated a new mouse model in which Foxm1 was specifically deleted from cardiomyocytes (Nkx2.5-Cre/Foxm1(fl/f) mice). Deletion of Foxm1 from cardiomyocytes was sufficient to disrupt heart morphogenesis and induce embryonic lethality in late gestation. Nkx2.5-Cre/Foxm1(fl/fl) hearts were dilated with thinning of the ventricular walls and interventricular septum, as well as disorganization of the myocardium which culminated in cardiac fibrosis and decreased capillary density. Cardiomyocyte proliferation was diminished in Nkx2.5-Cre/Foxm1(fl/fl) hearts owing to altered expression of multiple cell cycle regulatory genes, such as Cdc25B, Cyclin B(1), Plk-1, nMyc and p21(cip1). In addition, Foxm1 deficient hearts displayed reduced expression of CaMKIIδ, Hey2 and myocardin, which are critical mediators of cardiac function and myocardial growth. Our results indicate that Foxm1 expression in cardiomyocytes is critical for proper heart development and required for cardiomyocyte proliferation and myocardial growth.

Calcineurin is a widely expressed and highly conserved Ser/Thr phosphatase. Calcineurin is inhibited by the immunosuppressant drug cyclosporine A (CsA) or tacrolimus (FK506). The critical role of CsA/FK506 as an immunosuppressant following transplantation surgery provides a strong incentive to understand the phosphatase calcineurin. Here we uncover a novel regulatory pathway for cyclic AMP (cAMP) signaling by the phosphatase calcineurin which is also evolutionarily conserved in Caenorhabditis elegans. We found that calcineurin binds directly to and inhibits the proteosomal degradation of cAMP-hydrolyzing phosphodiesterase 4D (PDE4D). We show that ubiquitin conjugation and proteosomal degradation of PDE4D are controlled by a cullin 1-containing E(3) ubiquitin ligase complex upon dual phosphorylation by casein kinase 1 (CK1) and glycogen synthase kinase 3beta (GSK3beta) in a phosphodegron motif. Our findings identify a novel signaling process governing G-protein-coupled cAMP signal transduction-opposing actions of the phosphatase calcineurin and the CK1/GSK3beta protein kinases on the phosphodegron-dependent degradation of PDE4D. This novel signaling system also provides unique functional insights into the complications elicited by CsA in transplant patients.

The source of Ca2+ to activate pathological hypertrophy is not thought to involve the [Ca2+] that activates contraction. We hypothesize that Ca2+ influx through a subpopulation of L-type Ca2+ channels (CaV1.2; ICa,L) localized in caveolin (Cav) containing membrane signaling microdomains locally activates calcium/calmodulin and calcineurin-mediated NFAT nuclear translocation to induce hypertrophy. Rem-GTPase is known to inhibit CaV1.2 which requires a membrane association c-terminal. We truncated the c-terminal of Rem (Rem1-265) which eliminated CaV1.2 inhibition and fused Rem1-265 to a canonical caveolin binding domain to create Rem1-265-Cav. Results: Adenoviral-mediated expression of normal Rem in adult feline myocytes almost fully eliminated ICa,L while non-membrane targeted Rem1-265 had no significant effect on ICa,L. Rem1-265-Cav exhibited a small inhibition (less than 15%) of the normal ICa,L . Myocytes expressing Rem1-265-Cav responded normally to isoproterenol while those infected with normal Rem failed to respond. Myocytes infected with normal Rem had markedly reduced fractional shortening (2.4+/-0.7% resting cell length) while those infected with the truncated Rem1-265 (7.8+/-1.8) and Rem1-265-Cav (5.9+/-1.4) had contractions not significantly smaller than controls (7.4+/-2.1). Sucrose density gradient experiments revealed that Rem1-265-Cav cosedimented with caveolin-3 enriched low-density fractions. These experiments suggest Rem1-265-Cav inhibits caveolin-associated CaV1.2. To investigate the effects of our Rem constructs on NFAT nuclear translocation (hypertrophic signaling) myocytes were also infected with NFAT-GFP. Bath [Ca2+] was elevated to induce NFAT nuclear translocation measured by nuclear to cytoplasmic NFAT-GFP ratio. Normal Rem fully inhibited [Ca2+]-induced nuclear NFAT translocation and truncated Rem1-265 had no effect. Rem1-265-Cav inhibited more than 80% of [Ca2+]-induced NFAT nuclear translocation. Conclusion: These results suggest that CaV1.2 within Cav3 signaling microdomains is a major source of hypertrophic [Ca2+] signaling and it can be blocked with little or no effect on excitation-contraction coupling.

Previous studies have argued that enhanced activity of the epidermal growth factor receptor (EGFR) and the mitogen-activated protein kinase (MAPK) pathway can promote tumor cell survival in response to cytotoxic insults. In this study, we examined the impact of MAPK signaling on the survival of primary hepatocytes exposed to low concentrations of deoxycholic acid (DCA, 50 μM). Treatment of hepatocytes with DCA caused MAPK activation, which was dependent upon ligand independent activation of EGFR, and downstream signaling through Ras and PI3kinase. Neither inhibition of MAPK signaling alone by MEK1/2 inhibitors, nor exposure to DCA alone, enhanced basal hepatocyte apoptosis, whereas inhibition of DCA-induced MAPK activation caused ∼25% apoptosis within 6 h. Similar data were also obtained when either dominant negative EGFR-CD533 or dominant negative Ras N17 were used to block MAPK activation. DCA-induced apoptosis correlated with sequential cleavage of procaspase 8, BID, procaspase 9, and procaspase 3. Inhibition of MAPK potentiated bile acid-induced apoptosis in hepatocytes with mutant FAS-ligand, but did not enhance in hepatocytes that were null for FAS receptor expression. These data argues that DCA is causing ligand independent activation of the FAS receptor to stimulate an apoptotic response, which is counteracted by enhanced ligand-independent EGFR/MAPK signaling. In agreement with FAS-mediated cell killing, inhibition of caspase function with the use of dominant negative Fas-associated protein with death domain, a caspase 8 inhibitor (Ile-Glu-Thr-Asp-p-nitroanilide [IETD]) or dominant negative procaspase 8 blocked the potentiation of bile acid-induced apoptosis. Inhibition of bile acid-induced MAPK signaling enhanced the cleavage of BID and release of cytochrome cfrom mitochondria, which were all blocked by IETD. Despite activation of caspase 8, expression of dominant negative procaspase 9 blocked procaspase 3 cleavage and the potentiation of DCA-induced apoptosis. Treatment of hepatocytes with DCA transiently increased expression of the caspase 8 inhibitor proteins c-FLIP-Sand c-FLIP-Lthat were reduced by inhibition of MAPK or PI3kinase. Constitutive overexpression of c-FLIP-sabolished the potentiation of bile acid-induced apoptosis. Collectively, our data argue that loss of DCA-induced EGFR/Ras/MAPK pathway function potentiates DCA-stimulated FAS-induced hepatocyte cell death via a reduction in the expression of c-FLIP isoforms.

Hypertrophy is a basic cellular response to a variety of stressors and growth factors, and has been best characterized in myocytes. Pathologic hypertrophy of cardiac myocytes leads to heart failure, a major cause of death and disability in the developed world. Several cytosolic signaling pathways have been identified that transduce prohypertrophic signals, but to date, little work has focused on signaling pathways that might negatively regulate hypertrophy. Herein, we report that glycogen synthase kinase-3beta (GSK-3beta), a protein kinase previously implicated in processes as diverse as development and tumorigenesis, is inactivated by hypertrophic stimuli via a phosphoinositide 3-kinase-dependent protein kinase that phosphorylates GSK-3beta on ser 9. Using adenovirus-mediated gene transfer of GSK-3beta containing a ser 9 to alanine mutation, which prevents inactivation by hypertrophic stimuli, we demonstrate that inactivation of GSK-3beta is required for cardiomyocytes to undergo hypertrophy. Furthermore, our data suggest that GSK-3beta regulates the hypertrophic response, at least in part, by modulating the nuclear/cytoplasmic partitioning of a member of the nuclear factor of activated T cells family of transcription factors. The identification of GSK-3beta as a transducer of antihypertrophic signals suggests that novel therapeutic strategies to treat hypertrophic diseases of the heart could be designed that target components of the GSK-3 pathway.

Increased L type Ca channel (LTCC) activity was proposed to induce Ca dependent hypertrophy and heart failure by activating CaMK and calcineurin (CaN) signaling. Previous experiments showed that upregulation of LTCC activity induced profound cardiomyopathy. However, the enhanced Ca influx did not activate CaN or hypertrophy; but led to necrotic myocyte death. We hypothesized that reducing LTCC by genetic deletion of 50% of Cav1.2 would be cardioprotective like β-adrenergic receptor or LTCC blockers. Surprisingly, this deletion enhanced disease, CaN activation and hypertrophy. These myocytes with reduced Ca current exhibited significantly increased diastolic Ca. To further address these surprising findings we analyzed the Ca dynamics of mice with a standard null allele crossed with a cardiac specific flox allele (α1cf/-Cre), which led to an even greater deletion of the α1c protein in the heart. Here we measured Ca handling dynamics in α1cf/-Cre mice. Twitch amplitudes were significantly reduced (50%) and relaxation time was prolonged (16%). SR Ca load was reduced significantly (∼25%). These findings may be explained by increased diastolic SR Ca leak that we measured in myocytes from α1cf/-Cre mice. Thus, the deletion of the α1c protein in mouse cardiac myocytes leads to an increased basal [Ca]i by increasing Ca leak from the SR. Interestingly, the CaMKII inhibitor KN93 completely reversed the effects of the reduced LTCC: twitch amplitude and relaxation as well as SR Ca load were rescued. These results suggest that resting [Ca]i and the gain in ECC are increased to maintain contractility. This increase might drive hypertrophy by activating CaN and CaMKII.

Increases in Ca2+ influx through Cav1.2 has been observed in cardiovascular disease (CVD) and associated with cardiac arrhythmias. Methods: To mimic the enhanced Cav1.2 activity in CVD, we overexpressed the Cav1.2 β2a subunit in a transgenic mouse model. In-vivo ECGs, ion currents and intracellular Ca2+ were measured in transgenic (TG) and control (CTR) mice. Results: ICaL was greater in TG myocytes (23.9±2.5pA/pF, CTR 13.8±1.6pA/pF). TG mice had enhanced cardiac performance (EF: TG 72.7±1.3%, CTR 66.7±1.5%) but died suddenly (TG 50% vs CTR 100% alive at 6 months), suggesting cardiac arrhythmias. In conscious mice, there was no difference in heart rate in CTR (571±29bpm) and TG (541±24bpm) mice but the QT interval was significantly shorter in TG (44.0±5.5ms, CTR 58.2±3.4ms) mice. Second degree AV block and ectopic premature ventricular beats were observed in all 4 TG mice but not in CTR mice. In anesthetized mice, there was no difference in heart rate (CTR 513±20bpm, TG 526±13bpm) but the PR interval (CTR 32.4±1.4ms, TG 49.8±6.2ms) and QRS duration (CTR 11.4±0.8ms, TG 14.5±0.8ms) were significantly prolonged in TG mice, indicating conduction defects. A significantly greater % of TG myocytes (28.5%) had early (EADs) and delayed (DADs) afterdepolarizations than CTR (0.0%) due to enhanced SR load (caffeine spritz and Fluo-4 F/F0: TG 4.7±0.4 vs. CTR 3.2±0.3) and INCX (TG 2.15±0.6pA/pF vs CTR 1.12±0.3pA/pF at +60mV). However, action potential duration (APD) was significantly shorter in TG myocytes (APD90%: 40.0±5.7ms vs. CTR: 100.6±15.2ms) resulting from an increase of Ito (TG vs. CTR: 60.2±0.8pA/pF vs. 18.7±3.0pA/pF at +50mV). Conclusion: Persistent increases in Ca2+ influx through Cav1.2 cause both conduction disturbances and SR Ca overload, and induce cardiac arrhythmias with shortened APDs and QT intervals.